Increasing Resolution in Live Cell Microscopy by Structured Illumination (SIM)

Abstract

In the context of various approaches to super-resolution microscopy, structured illumination microscopy (SIM) offers several advantages: it needs rather low light doses (with a low risk of phototoxicity or photobleaching), is comparably fast and flexible concerning the use of microscopes, objective lenses and cameras, and has potential for 3D imaging. This paper describes an experimental setup for SIM with first diffraction orders of a spectral light modulator (SLM) creating an interference pattern in two dimensions. We kept this system rather compact with a comparably large illuminated object field, validated it with nano-beads and applied it further to living cells for imaging the cytoskeleton, mitochondria or cell nuclei with a resolution slightly above 100 nm. Its advantages, challenges and limitations—concerning cameras, acquisition time, depth of imaging, light exposure, and combining it with further super-resolving methods—are discussed.

Super-Resolution Live Cell Microscopy of Membrane-Proximal Fluorophores

“…The experimental technique is based on a setup for SIM reported previously [ 21 ] and is summarized as follows: Illumination occurs by an expanded argon ion laser beam (λ 0 = 488 nm). Interference patterns resulting from the first diffraction orders of the SLM (ferroelectric LCOS, SXGA-3DM, Forth Dimension Displays, UK) are recorded for three phases (0, 2π/3, 4π/3) and three rotation angles (0°, 60°, and 120°) in order to obtain an optical transfer function (OTF) of rotational symmetry.…”

“…The present manuscript describes an application of Structured Illumination Microscopy (SIM). It shows how a custom-built experimental setup for SIM [ 21 ] can be extended for TIRF-SIM using a high-aperture microscope objective lens. The focal lengths of the lenses in the illumination beam have to be adapted according to Figure 1 , while further components—e.g., λ/4 plate, pinhole, and “pizza polarizer”—can be maintained for the selection of the first diffraction orders of the SLM in three directions with the appropriate (azimuthal) polarizations.…”

“… Imaging in SIM and TIRF-SIM (schematic) with a spatial light modulator SLM, telescope lenses L 1 and L 2 , tube lens TL, and objective lens OL. The illumination of the SLM by the argon ion laser (488 nm) as well as further components (λ/4 plate, pinhole plate, “pizza polarizer”, dichroic mirror, and camera, as reported previously [ 21 ]) are omitted. The beam alignment is identical for the SIM and TIRF-SIM apart from the objective lens OL, resulting in different grating constants Δ’ in the plane of the sample (d = grating constant of the SLM; Δ = grating constant of the interference pattern).…”

“…Here, we present a robust SLM-based SIM technique, which, due to its linear arrangement and to a minimum of optical components in comparison to previous systems (e.g., [ 18 , 19 , 20 ]), is easy to perform and to implement, and which is finally cost-effective. Our setup represents an improvement of a previously reported system [ 21 ], modified for TIRF microscopy as well as for switching between SIM und TIRF-SIM.…”

High-speed, multicolor, structured illumination microscopy using a hexagonal single mode fiber array

“…For systems based on Spatial Light Modulators (SLMs) and Digital Micromirror Devices (DMDs), if only 2D SIM is being pursued, various groups have used a quarter waveplate to generate circularly polarized light after the diffractive device (25). The diffracted beams are then sent through a "pizza" polarizer, which is a multi-segmented polarizer cut into wedges (26,27). Each wedge is aligned on a circle such that the polarization axis is perpendicular to the radius, creating an S polarized interference pattern at the sample.…”